Cancer-linked gene as target for chemotherapy

ABSTRACT

Cancer-linked gene sequences, and derived amino acid sequences, are disclosed along with processes for assaying potential antitumor agents based on their modulation of the expression of these cancer-linked genes. Also disclosed are antibodies that react with the disclosed polypeptides and methods of using the antibodies to treat cancerous conditions, such as by using the antibody to target cancerous cells in vivo for purposes of delivering therapeutic agents thereto. Also described are methods of diagnosing using the gene sequences.

This application claims priority of U.S. Provisional Patent Application60/386,793, filed 7 Jun. 2002, the disclosure of which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of screening cancer-linkedgenes and expression products for involvement in the cancer initiationand facilitation process as a means of cancer diagnosis as well as theuse of such genes for screening potential anti-cancer agents, includingsmall organic compounds and other molecules, and development oftherapeutic agents.

BACKGROUND OF THE INVENTION

Cancer-linked genes are valuable in that they indicate geneticdifferences between cancer cells and normal cells, such as where a geneis expressed in a cancer cell but not in a non-cancer cell, or wheresaid gene is over-expressed or expressed at a higher level in a canceras opposed to normal or non-cancer cell. In addition, the expression ofsuch a gene in a normal cell but not in a cancer cell, especially of thesame type of tissue, can indicate important functions in the cancerousprocess. For example, screening assays for novel drugs are based on theresponse of model cell based systems in vitro to treatment with specificcompounds. Such genes are also useful in the diagnosis of cancer and theidentification of a cell as cancerous. Gene activity is readily measuredby measuring the rate of production of gene products, such as RNAs andpolypeptides encoded by such genes. Where genes encode cell surfaceproteins, appearance of, or alterations in, such proteins, as cellsurface markers, are an indication of neoplastic activity. Some suchscreens rely on specific genes, such as oncogenes (or gene mutations).In accordance with the present invention, a cancer-linked gene has beenidentified and its putative amino acid sequence worked out. Such gene isuseful in the diagnosing of cancer, the screening of anticancer agentsand the treatment of cancer using such agents, especially in that thesegenes encode polypeptides that can act as markers, such as cell surfacemarkers, thereby providing ready targets for anti-tumor agents such asantibodies, preferably antibodies complexed to cytotoxic agents,including apoptotic agents.

BRIEF SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided herein acancer specific gene, linked especially to breast or endometrium cancer,or otherwise involved in the cancer initiating and facilitating processand the derived amino acid sequence thereof, including a number ofdifferent transcripts derived from said gene.

In one aspect, the present invention relates to a process foridentifying an agent that modulates the activity of a cancer-relatedgene comprising:

(a) contacting a compound with a cell containing a gene that correspondsto a polynucleotide having a sequence selected from the group consistingof SEQ ID NO: 1, 2, 3 and 4 and under conditions promoting theexpression of said gene; and

(b) detecting a difference in expression of said gene relative to whensaid compound is not present

thereby identifying an agent that modulates the activity of acancer-related gene.

In various embodiments of such a process, the cell is a cancer cell andthe difference in expression is a decrease in expression. Suchpolynucleotides may also include those that have sequences identical toSEQ ID NO: 1, 2, 3 and 4.

In another aspect, the present invention relates to a process foridentifying an anti-neoplastic agent comprising contacting a cellexhibiting neoplastic activity with a compound first identified as acancer related gene modulator using an assay process disclosed hereinand detecting a decrease in said neoplastic activity after saidcontacting compared to when said contacting does not occur. Suchneoplastic activity may include accelerated cellular replication and/ormetastasis, and the decrease in neoplastic activity preferably resultsfrom the death of the cell, or senescence, terminal differentiation orgrowth inhibition.

The present invention also relates to a process for identifying ananti-neoplastic agent comprising administering to an animal exhibiting acancer condition an effective amount of an agent first identifiedaccording to a process of one of one of the assays disclosed accordingto the invention and detecting a decrease in said cancerous condition.

The present invention further relates to a process for determining thecancerous status of a cell, comprising determining an increase in thelevel of expression in said cell of at least one gene that correspondsto a polynucleotide having a sequence selected from the group consistingof SEQ ID NO: 1, 2, 3 and 4 wherein an elevated expression relative to aknown non-cancerous cell indicates a cancerous state or potentiallycancerous state. Such elevated expression may be due to an increasedcopy number.

The present invention additionally relates to an isolated polypeptide,encoded by one of the polynucleotide transcripts disclosed herein,comprising an amino acid sequence homologous to an amino acid sequenceselected from the group consisting of SEQ ID NO: 5 and 6 wherein anydifference between said amino acid sequence and the sequence of SEQ IDNO: 5 and 6 is due solely to conservative amino acid substitutions andwherein said isolated polypeptide comprises at least one immunogenicfragment. In a preferred embodiment, the present invention encompassesan isolated polypeptide comprising an amino acid sequence homologous toan amino acid sequence selected from the group consisting of SEQ ID NO:5 and 6.

The present invention also relates to an antibody that reacts with apolypeptide as disclosed herein, preferably a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 5and 6. Such an antibody may be polyclonal, monoclonal, recombinant orsynthetic in origin.

In one such embodiment, said antibody is associated, either covalentlyor non-covalently, with a cytotoxic agent, for example, an apoptoticagent. Thus, the present invention relates to an immunoconjugatecomprising an antibody of the invention and a cytotoxic agent.

The present invention also relates to a process for treating cancercomprising contacting a cancerous cell with an agent having activityagainst an expression product encoded by a gene sequence selected fromthe group consisting of SEQ ID NO: 1, 2, 3 and 4. In one suchembodiment, the cancerous cell is contacted in vivo. In another suchembodiment, said agent has affinity for said expression product. In apreferred embodiment, such agent is an antibody disclosed herein, suchas an antibody that is specific or selective for, or otherwise reactswith, a polypeptide of the invention. In a preferred embodiment, theexpression product is a polypeptide incorporating an amino acid sequenceselected from SEQ ID NO: 5 and 6.

The present invention further encompasses an immunogenic compositioncomprising a polypeptide disclosed herein, as well as compositionsformed using antibodies specific for these polypeptides.

The present invention is also directed to uses of such compositions.Such uses include a method for treating cancer in an animal afflictedtherewith comprising administering to said animal an amount of animmunogenic composition of one or more of the polypeptides disclosedherein where such amount is an amount sufficient to elicit theproduction of cytotoxic T lymphocytes specific for a polypeptide of theinvention, preferably a polypeptide incorporating a sequence of SEQ IDNO: 5 and 6. In a preferred embodiment, the animal to be so treated is ahuman patient.

DEFINITIONS

As used herein, the terms “portion,” “segment,” and “fragment,” whenused in relation to polypeptides, refer to a continuous sequence ofresidues, such as amino acid residues, which sequence forms a subset ofa larger sequence. For example, if a polypeptide were subjected totreatment with any of the common endopeptidases, such as trypsin orchymotrypsin, the oligopeptides resulting from such treatment wouldrepresent portions, segments or fragments of the starting polypeptide.When used in relation to a polynucleotides, such terms refer to theproducts produced by treatment of said polynucleotides with any of thecommon endonucleases.

As used herein, the term “isolated” means that the material is removedfrom its original environment (e.g., the natural environment if it isnaturally occurring). It could also be produced recombinantly andsubsequently purified. For example, a naturally-occurring polynucleotideor polypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides, for example, those prepared recombinantly, could bepart of a vector and/or such polynucleotides or polypeptides could bepart of a composition, and still be isolated in that such vector orcomposition is not part of its natural environment. In one embodiment ofthe present invention, such isolated, or purified, polypeptide is usefulin generating antibodies for practicing the invention, or where saidantibody is attached to a cytotoxic or cytolytic agent, such as anapoptotic agent.

The term “percent identity” or “percent identical,” when referring to asequence, means that a sequence is compared to a claimed or describedsequence after alignment of the sequence to be compared (the “ComparedSequence”) with the described or claimed sequence (the “ReferenceSequence”). The Percent Identity is then determined according to thefollowing formula:Percent Identity=100[1−(C/R)]wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence wherein (i) each base oramino acid in the Reference Sequence that does not have a correspondingaligned base or amino acid in the Compared Sequence and (ii) each gap inthe Reference Sequence and (iii) each aligned base or amino acid in theReference Sequence that is different from an aligned base or amino acidin the Compared Sequence, constitutes a difference; and R is the numberof bases or amino acids in the Reference Sequence over the length of thealignment with the Compared Sequence with any gap created in theReference Sequence also being counted as a base or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which thehereinabove calculated Percent Identity is less than the specifiedPercent Identity.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

In accordance with the present invention, the term “DNA segment” or “DNAsequence” refers to a DNA polymer, in the form of a separate fragment oras a component of a larger DNA construct, which has been derived fromDNA isolated at least once in substantially pure form, i.e., free ofcontaminating endogenous materials and in a quantity or concentrationenabling identification, manipulation, and recovery of the segment andits component nucleotide sequences by standard biochemical methods, forexample, using a cloning vector. Such segments are provided in the formof an open reading frame uninterrupted by internal nontranslatedsequences, or introns, which are typically present in eukaryotic genes.Sequences of non-translated DNA may be present downstream from the openreading frame, where the same do not interfere with manipulation orexpression of the coding regions.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene. The coding region can be from anormal, mutated or altered gene, or can even be from a DNA sequence, orgene, wholly synthesized in the laboratory using methods well known tothose of skill in the art of DNA synthesis.

In accordance with the present invention, the term “nucleotide sequence”refers to a heteropolymer of deoxyribonucleotides. Generally, DNAsegments encoding the proteins provided by this invention are assembledfrom cDNA fragments and short oligonucleotide linkers, or from a seriesof oligonucleotides, to provide a synthetic gene which is capable ofbeing expressed in a recombinant transcriptional unit comprisingregulatory elements derived from a microbial, eukaryotic or viraloperon.

The term “expression product” means that polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “active fragment,” when referring to a coding sequence, means aportion comprising less than the complete coding region whose expressionproduct retains essentially the same biological function or activity asthe expression product of the complete coding region.

The term “primer” means a short nucleic acid sequence that is pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription. The term “enhancer” refers to aregion of DNA that, when present and active, has the effect ofincreasing expression of a different DNA sequence that is beingexpressed, thereby increasing the amount of expression product formedfrom said different DNA sequence.

The term “open reading frame (ORF)” means a series of triplets codingfor amino acids without any termination codons and is a sequence(potentially) translatable into protein.

As used herein, reference to a “DNA sequence” includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence.

As used herein, “corresponding genes” refers to genes that encode an RNAthat is at least 90% identical, preferably at least 95% identical, mostpreferably at least 98% identical, and especially identical, to an RNAencoded by one of the nucleotide sequences disclosed herein (i.e., SEQID NO: 1, 2, 3 and 4). Such genes will also encode the same polypeptidesequence as any of the sequences disclosed herein, preferably SEQ ID NO:1, 2, 3 and 4, but may include differences in such amino acid sequenceswhere such differences are limited to conservative amino acidsubstitutions, such as where the same overall three dimensionalstructure, and thus the same antigenic character, is maintained. Thus,amino acid sequences may be within the scope of the present inventionwhere they react with the same antibodies that react with polypeptidescomprising the sequences of SEQ ID NO: 5 and 6. A “corresponding gene”includes splice variants thereof.

The genes identified by the present disclosure are considered“cancer-related” genes, as this term is used herein, and include genesexpressed at higher levels (due, for example, to elevated rates ofexpression, elevated extent of expression or increased copy number) incancer cells relative to expression of these genes in normal (i.e.,non-cancerous) cells where said cancerous state or status of test cellsor tissues has been determined by methods known in the art, such as byreverse transcriptase polymerase chain reaction (RT-PCR) as described inthe Examples herein. In specific embodiments, this relates to the geneswhose sequences correspond to the sequences of SEQ ID NO: 1, 2, 3 and 4.

As used herein, the term “conservative amino acid substitutions” aredefined herein as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

-   -   Asp, Asn, Glu, Gln;

III. Polar, positively charged residues:

-   -   His, Arg, Lys;

IV. Large, aliphatic, nonpolar residues:

-   -   Met Leu, Ile, Val, Cys

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp

DETAILED SUMMARY OF THE INVENTION

The present invention relates to processes for utilizing a nucleotidesequence for a cancer-linked gene, polypeptides encoded by suchsequences and antibodies reactive with such polypeptides in methods oftreating and diagnosing cancer, preferably breast or endometrium cancer,and in carrying out screening assays for agents effective in reducingthe activity of cancer-linked genes and thereby treating a cancerouscondition.

The polypeptides disclosed herein incorporate various polynucleotidetranscripts (SEQ ID NO: 1, 2, 3 and 4) and the derived amino acidsequence (SEQ ID NO: 5 and 6) from said transcripts are available astargets for chemotherapeutic agents, especially anti-cancer agents,including antibodies specific for said polypeptides.

The cancer-related polynucleotide sequences disclosed herein correspondto gene sequences whose expression is indicative of the cancerous statusof a given cell. Such sequences are substantially identical to SEQ IDNO: 1, 2, 3 and 4, which represent different transcripts identified fromthe GenBank EST database and which exhibit cancer-specific expression.The polynucleotides of the invention are those that correspond to asequence of SEQ ID NO: 1, 2, 3 and 4. Such sequences have been searchedwithin the GenBank database, especially the EST database, with thefollowing results:

-   Type: cell-surface tumor antigen therapeutic antibody target-   Tissue: breast and endometrium-   AffyFragment-ID(s): 125026, 142673 hypothetical protein FLJ22418-   Accession(s): AA075632, AI799522-   Unigene cluster-ID(s): Hs.36563-   Chromosomal location: 1

The nucleotides and polypeptides, as gene products, used in theprocesses of the present invention may comprise a recombinantpolynucleotide or polypeptide, a natural polynucleotide or polypeptide,or a synthetic polynucleotide or polypeptide, or a recombinantpolynucleotide or polypeptide.

Fragments of such polynucleotides and polypeptides as are disclosedherein may also be useful in practicing the processes of the presentinvention. For example, a fragment, derivative or analog of thepolypeptide (SEQ ID NO: 5 and 6) may be (i) one in which one or more ofthe amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide (such as a histidine hexapeptide) or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

In another aspect, the present invention relates to an isolatedpolypeptide, including a purified polypeptide, comprising an amino acidsequence at least 90% identical to the amino acid sequence of SEQ ID NO:5 and/or 6. In preferred embodiments, said isolated polypeptidecomprises an amino acid sequence having sequence identity of at least95%, preferably at least about 98%, and especially is identical to, thesequence of SEQ ID NO: 5 and/or 6. The present invention also includesisolated active fragments of such polypeptides where said fragmentsretain the biological activity of the polypeptide or where such activefragments are useful as specific targets for cancer treatment,prevention or diagnosis. Thus, the present invention relates to anypolypeptides, or fragments thereof, with sufficient sequence homology tothe sequences disclosed herein as to be useful in the production ofantibodies that react with (i.e., are selective or specific for) thepolypeptides of SEQ ID NO: 5 and 6 so as to be useful in targeting cellsthat exhibit such polypeptides, or fragments, on their surfaces, therebyproviding targets for such antibodies and therapeutic agents associatedwith such antibodies.

The polynucleotides and polypeptides useful in practicing the processesof the present invention may likewise be obtained in an isolated orpurified form. In addition, the polypeptide disclosed herein as beinguseful in practicing the processes of the invention are believed to besurface proteins present on cells, such as cancerous cells. Preciselyhow such cancer-linked proteins are used in the processes of theinvention may thus differ depending on the therapeutic approach used.For example, cell-surface proteins, such as receptors, are desirabletargets for cytotoxic antibodies that can be generated against thepolypeptides disclosed herein.

The sequence information disclosed herein, as derived from the GenBanksubmissions, can readily be utilized by those skilled in the art toprepare the corresponding full-length polypeptide by peptide synthesis.The same is true for either the polynucleotides or polypeptidesdisclosed herein for use in the methods of the invention.

The present invention relates to an isolated polypeptide, encoded by oneof the polynucleotide transcripts disclosed herein, comprising an aminoacid sequence homologous to an amino acid sequence selected from thegroup consisting of SEQ ID NO: 5 and 6, wherein any difference betweenamino acid sequence in the isolated polypeptide and the sequence of SEQID NO: 5 and 6 is due solely to conservative amino acid substitutionsand wherein said isolated polypeptide comprises at least one immunogenicfragment. In a preferred embodiment, the present invention encompassesan isolated polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 5 and 6.

Methods of producing recombinant cells and vectors useful in preparingthe polynucleotides and polypeptides disclosed herein are well known tothose skilled in the molecular biology art. See, for example, Sambrook,et al., Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor, N.Y., (1989), Wu et al., Methods in Gene Biotechnology(CRC Press, New York, N.Y., 1997), and Recombinant Gene ExpressionProtocols, in Methods in Molecular Biology, Vol. 62, (Tuan, ed., HumanaPress, Totowa, N.J., 1997), the disclosures of which are herebyincorporated by reference.

In one aspect, the present invention relates to a process foridentifying an agent that modulates the activity of a cancer-relatedgene comprising:

(a) contacting a compound with a cell containing a gene that correspondsto a polynucleotide having a sequence selected from the group consistingof SEQ ID NO: 1, 2, 3 and 4 and under conditions promoting theexpression of said gene; and

(b) detecting a difference in expression of said gene relative to whensaid compound is not present

thereby identifying an agent that modulates the activity of acancer-related gene.

In specific embodiments of such process the cell is a cancer cell andthe difference in expression is a decrease in expression. Suchpolynucleotides may also include those that have sequences identical toSEQ ID NO: 1, 2, 3 and 4.

In another aspect, the present invention relates to a process foridentifying an anti-neoplastic agent comprising contacting a cellexhibiting neoplastic activity with a compound first identified as acancer related gene modulator using an assay process disclosed hereinand detecting a decrease in said neoplastic activity after saidcontacting compared to when said contacting does not occur. Suchneoplastic activity may include accelerated cellular replication and/ormetastasis, and the decrease in neoplastic activity preferably resultsfrom the death of the cell.

The present invention also relates to a process for identifying ananti-neoplastic agent comprising administering to an animal exhibiting acancer condition an effective amount of an agent first identifiedaccording to a process of one of one of the assays disclosed accordingto the invention and detecting a decrease in said cancerous condition.

In specific embodiments of the present invention, the genes useful forthe invention comprise genes that correspond to polynucleotides having asequence selected from SEQ ID NO: 1, 2, 3 and 4, or may comprise thesequence of any of the polynucleotides disclosed herein (where thelatter are cDNA sequences).

In accordance with the present invention, such assays rely on methods ofdetermining the activity of the gene in question. Such assays areadvantageously based on model cellular systems using cancer cell lines,primary cancer cells, or cancerous tissue samples that are maintained ingrowth medium and treated with compounds at a single concentration or ata range of concentrations. At specific times after treatment, cellularRNAs are conveniently isolated from the treated cells or tissues, whichRNAs are indicative of expression of selected genes. The cellular RNA isthen divided and subjected to differential analysis that detects thepresence and/or quantity of specific RNA transcripts, which transcriptsmay then be amplified for detection purposes using standardmethodologies, such as, for example, reverse transcriptase polymerasechain reaction (RT-PCR), etc. The presence or absence, or concentrationlevels, of specific RNA transcripts are determined from thesemeasurements. The polynucleotide sequences disclosed herein are readilyused as probes for the detection of such RNA transcripts and thus themeasurement of gene activity and expression.

The polynucleotides of the invention can include fully operational geneswith attendant control or regulatory sequences or merely apolynucleotide sequence encoding the corresponding polypeptide or anactive fragment or analog thereof.

Because expression of the polynucleotide sequences disclosed herein arespecific to the cancerous state, useful gene modulation is downwardmodulation, so that, as a result of exposure to an antineoplastic agentidentified by the screening assays herein, the corresponding gene of thecancerous cell is expressed at a lower level (or not expressed at all)when exposed to the agent as compared to the expression when not exposedto the agent. For example, the gene sequences disclosed herein (SEQ IDNO: 1, 2, 3 and 4) correspond to a gene expressed at a higher level incells of breast or endometrium cancer than in normal breast orendometrium cells. Thus, where said chemical agent causes this gene ofthe tested cell to be expressed at a lower level than the same genes ofthe reference, this is indicative of downward modulation and indicatesthat the chemical agent to be tested has anti-neoplastic activity.

In carrying out the assays disclosed herein, relative antineoplasticactivity may be ascertained by the extent to which a given chemicalagent modulates the expression of genes present in a cancerous cell.Thus, a first chemical agent that modulates the expression of a geneassociated with the cancerous state (i.e., a gene corresponding to oneor more of the polynucleotide transcripts disclosed herein) to a largerdegree than a second chemical agent tested by the assays of theinvention is thereby deemed to have higher, or more desirable, or moreadvantageous, anti-neoplastic activity than said second chemical agent.

The gene expression to be measured is commonly assayed using RNAexpression as an indicator. Thus, the greater the level of RNA (forexample, messenger RNA or mRNA) detected the higher the level ofexpression of the corresponding gene. Thus, gene expression, eitherabsolute or relative, is determined by the relative expression of theRNAs encoded by such genes.

RNA may be isolated from samples in a variety of ways, including lysisand denaturation with a phenolic solution containing a chaotropic agent(e.g., trizol) followed by isopropanol precipitation, ethanol wash, andresuspension in aqueous solution; or lysis and denaturation followed byisolation on solid support, such as a Qiagen resin and reconstitution inaqueous solution; or lysis and denaturation in non-phenolic, aqueoussolutions followed by enzymatic conversion of RNA to DNA templatecopies.

Normally, prior to applying the processes of the invention, steady stateRNA expression levels for the genes, and sets of genes, disclosed hereinwill have been obtained. It is the steady state level of such expressionthat is affected by potential anti-neoplastic agents as determinedherein. Such steady state levels of expression are easily determined byany methods that are sensitive, specific and accurate. Such methodsinclude, but are in no way limited to, real time quantitative polymerasechain reaction (PCR), for example, using a Perkin-Elmer 7700 sequencedetection system with gene specific primer probe combinations asdesigned using any of several commercially available software packages,such as Primer Express software., solid support based hybridizationarray technology using appropriate internal controls for quantitation,including filter, bead, or microchip based arrays, solid support basedhybridization arrays using, for example, chemiluminescent, fluorescent,or electrochemical reaction based detection systems.

The gene expression indicative of a cancerous state need not becharacteristic of every cell of a given tissue. Thus, the methodsdisclosed herein are useful for detecting the presence of a cancerouscondition within a tissue where less than all cells exhibit the completepattern. Thus, for example, a selected gene corresponding to thesequence of SEQ ID NO: 1, may be found, using appropriate probes, eitherDNA or RNA, to be present in as little as 60% of cells derived from asample of tumorous, or malignant, tissue. In a highly preferredembodiment, such gene pattern is found to be present in at least 100% ofcells drawn from a cancerous tissue and absent from at least 100% of acorresponding normal, non-cancerous, tissue sample.

Expression of a gene may be related to copy number, and changes inexpression may be measured by determining copy number. Such change ingene copy number may be determined by determining a change in expressionof messenger RNA encoded by a particular gene sequence, especially thatof SEQ ID NO: 1, 2, 3 and 4. Also in accordance with the presentinvention, said gene may be a cancer initiating or facilitating gene. Incarrying out the methods of the present invention, a cancer facilitatinggene is a gene that, while not directly initiating tumor formation orgrowth, acts, such as through the actions of its expression product, todirect, enhance, or otherwise facilitate the progress of the cancerouscondition, including where such gene acts against genes, or geneexpression products, that would otherwise have the effect of decreasingtumor formation and/or growth.

Although the expression of a gene corresponding to a sequence of SEQ IDNO: 1, 2, 3 and 4 may be indicative of a cancerous status for a givencell, the mere presence of such a gene may not alone be sufficient toachieve a malignant condition and thus the level of expression of suchgene may also be a significant factor in determining the attainment of acancerous state. Thus, it becomes essential to also determine the levelof expression of a gene as disclosed herein, including substantiallysimilar sequences, as a separate means of diagnosing the presence of acancerous status for a given cell, groups of cells, or tissues, eitherin culture or in situ.

The level of expression of the polypeptides disclosed herein is also ameasure of gene expression, such as polypeptides having sequenceidentical, or similar to, any polypeptide encoded by a sequence of SEQID NO: 1, 2, 3 and 4, especially a polypeptide whose amino acid sequenceis the sequence of SEQ ID NO: 5 and 6.

In accordance with the foregoing, the present invention specificallycontemplates a method for determining the cancerous status of a cell tobe tested, comprising determining the level of expression in said cellof a gene that includes one of the nucleotide sequences selected fromthe sequences of SEQ ID NO: 1, 2, 3 and 4, including sequencessubstantially identical to said sequences, or characteristic fragmentsthereof, or the complements of any of the foregoing and then comparingsaid expression to that of a cell known to be non-cancerous whereby thedifference in said expression indicates that said cell to be tested iscancerous.

In accordance with the invention, although gene expression for a genethat includes as a portion thereof one of the sequences of SEQ ID NO: 1,2, 3 and 4, is preferably determined by use of a probe that is afragment of such nucleotide sequence, it is to be understood that theprobe may be formed from a different portion of the gene. Expression ofthe gene may be determined by use of a nucleotide probe that hybridizesto messenger RNA (mRNA) transcribed from a portion of the gene otherthan the specific nucleotide sequence disclosed herein.

It should be noted that there are a variety of different contexts inwhich genes have been evaluated as being involved in the cancerousprocess. Thus, some genes may be oncogenes and encode proteins that aredirectly involved in the cancerous process and thereby promote theoccurrence of cancer in an animal. In addition, other genes may serve tosuppress the cancerous state in a given cell or cell type and therebywork against a cancerous condition forming in an animal. Other genes maysimply be involved either directly or indirectly in the cancerousprocess or condition and may serve in an ancillary capacity with respectto the cancerous state. All such types of genes are deemed with those tobe determined in accordance with the invention as disclosed herein.Thus, the gene determined by said process of the invention may be anoncogene, or the gene determined by said process may be a cancerfacilitating gene, the latter including a gene that directly orindirectly affects the cancerous process, either in the promotion of acancerous condition or in facilitating the progress of cancerous growthor otherwise modulating the growth of cancer cells, either in vivo or exvivo. In addition, the gene determined by said process may be a cancersuppressor gene, which gene works either directly or indirectly tosuppress the initiation or progress of a cancerous condition. Such genesmay work indirectly where their expression alters the activity of someother gene or gene expression product that is itself directly involvedin initiating or facilitating the progress of a cancerous condition. Forexample, a gene that encodes a polypeptide, either wild or mutant intype, which polypeptide acts to suppress of tumor suppressor gene, orits expression product, will thereby act indirectly to promote tumorgrowth.

As noted previously, polynucleotides encoding the same proteins as anyof SEQ ID NO: 1, 2, 3 and 4, regardless of the percent identity of suchsequences, are also specifically contemplated by any of the methods ofthe present invention that rely on any or all of said sequences,regardless of how they are otherwise described or limited. Thus, anysuch sequences are available for use in carrying out any of the methodsdisclosed according to the invention. Such sequences also include anyopen reading frames, as defined herein, present within the sequence ofSEQ ID NO: 1, 2, 3 and 4.

Because a gene disclosed according to the invention “corresponds to” apolynucleotide having a sequence of SEQ ID NO: 1, 2, 3 and 4, said geneencodes an RNA (processed or unprocessed, including naturally occurringsplice variants and alleles) that is at least 90% identical, preferablyat least 95% identical, most preferably at least 98% identical to, andespecially identical to, an RNA that would be encoded by, or becomplementary to, such as by hybridization with, a polynucleotide havingthe indicated sequence. In addition, genes including sequences at least90% identical to a sequence selected from SEQ ID NO: 1, 2, 3 and 4,preferably at least about 95% identical to such a sequence, morepreferably at least about 98% identical to such sequence and mostpreferably comprising such sequence are specifically contemplated by allof the processes of the present invention. Sequences encoding the sameproteins as any of these sequences, regardless of the percent identityof such sequences, are also specifically contemplated by any of themethods of the present invention that rely on any or all of saidsequences, regardless of how they are otherwise described or limited.The polynucleotide sequences of the invention also include any openreading frames, as defined herein, present within any of the sequencesof SEQ ID NO: 1, 2, 3 and 4.

The sequences disclosed herein may be genomic in nature and thusrepresent the sequence of an actual gene, such as a human gene, or maybe a cDNA sequence derived from a messenger RNA (mRNA) and thusrepresent contiguous exonic sequences derived from a correspondinggenomic sequence, or they may be wholly synthetic in origin for purposesof practicing the processes of the invention. Because of the processingthat may take place in transforming the initial RNA transcript into thefinal mRNA, the sequences disclosed herein may represent less than thefull genomic sequence. They may also represent sequences derived fromribosomal and transfer RNAs. Consequently, the gene as present in thecell (and representing the genomic sequence) and the polynucleotidetranscripts disclosed herein, including cDNA sequences, may be identicalor may be such that the cDNAs contain less than the full genomicsequence. Such genes and cDNA sequences are still considered“corresponding sequences” (as defined elsewhere herein) because theyboth encode the same or related RNA sequences (i.e., related in thesense of being splice variants or RNAs at different stages ofprocessing). Thus, by way of non-limiting example only, a gene thatencodes an RNA transcript, which is then processed into a shorter mRNA,is deemed to encode both such RNAs and therefore encodes an RNAcomplementary to (using the usual Watson-Crick complementarity rules),or that would otherwise be encoded by, a cDNA (for example, a sequenceas disclosed herein). Thus, the sequences disclosed herein correspond togenes contained in the cancerous cells (here, breast or endometriumcancer) and are used to determine gene activity or expression becausethey represent the same sequence or are complementary to RNAs encoded bythe gene. Such a gene also includes different alleles and splicevariants that may occur in the cells used in the methods of theinvention, such as where recombinant cells are used to assay foranti-neoplastic agents and such cells have been engineered to express apolynucleotide as disclosed herein, including cells that have beenengineered to express such polynucleotides at a higher level than isfound in non-engineered cancerous cells or where such recombinant cellsexpress such polynucleotides only after having been engineered to do so.Such engineering includes genetic engineering, such as where one or moreof the polynucleotides disclosed herein has been inserted into thegenome of such cell or is present in a vector.

Such cells, especially mammalian cells, may also be engineered toexpress on their surfaces one or more of the polypeptides of theinvention for testing with antibodies or other agents capable of maskingsuch polypeptides and thereby removing the cancerous nature of the cell.Such engineering includes both genetic engineering, where the geneticcomplement of the cells is engineered to express the polypeptide, aswell as non-genetic engineering, whereby the cell has been physicallymanipulated to incorporate a polypeptide of the invention in its plasmamembrane, such as by direct insertion using chemical and/or other agentsto achieve this result.

In accordance with the foregoing, the present invention includesanti-cancer agents that are themselves either polypeptides, or smallchemical entities, that affect the cancerous process, includinginitiation, suppression or facilitation of tumor growth, either in vivoor ex vivo. Said cancer modulating agent will have the effect ofdecreasing gene expression.

The present invention thus also relates to a method for treating cancercomprising contacting a cancerous cell with an agent having activityagainst an expression product encoded by a gene or polynucleotidesequence as disclosed herein, such as one having, or corresponding to,the nucleotide sequence of SEQ ID NO: 1, 2, 3 and 4. The presentinvention also relates to a process for treating cancer comprisingcontacting a cancerous cell with an agent having activity against anexpression product encoded by a gene or polynucleotide sequencecorresponding to a sequence selected from the group consisting of SEQ IDNO: 1, 2, 3 and 4. In one such embodiment, the cancerous cell iscontacted in vivo. In another such embodiment, said agent has affinityfor said expression product. In a preferred embodiment, such agent is anantibody disclosed herein, such as an antibody that is specific orselective for, or otherwise reacts with, a polypeptide of the invention.In a preferred embodiment, the expression product is a polypeptideincorporating an amino acid sequence selected from SEQ ID NO: 5 and 6.

The present invention is also directed to such uses of the compositionsof polypeptides and antibodies disclosed herein. Such uses include aprocess for treating cancer in an animal afflicted therewith comprisingadministering to said animal an amount of an immunogenic composition ofone or more of the polypeptides disclosed herein where such amount if anamount sufficient to elicit the production of cytotoxic T lymphocytesspecific for a polypeptide of the invention, preferably a polypeptideincorporating a sequence of SEQ ID NO: 5 and 6. In a preferredembodiment, the animal to be so treated is a human patient.

The proteins encoded by the genes disclosed herein due to theirexpression, or elevated expression, in cancer cells, represent highlyuseful therapeutic targets for “targeted therapies” utilizing suchaffinity structures as, for example, antibodies coupled to somecytotoxic agent. In such methodology, it is advantageous that nothingneed be known about the endogenous ligands or binding partners for suchcell surface molecules. Rather, an antibody or equivalent molecule thatcan specifically recognize the cell surface molecule (which couldinclude an artificial peptide, a surrogate ligand, and the like) that iscoupled to some agent that can induce cell death or a block in cellcycling offers therapeutic promise against these proteins. Thus, suchapproaches include the use of so-called suicide “bullets” againstintracellular proteins. For example, monoclonal antibodies may readilyby produced by methods well known in the art, for example, the method ofKohler and Milstein (see: Nature, 256:495 (1975).

With the advent of methods of molecular biology and recombinanttechnology, it is now possible to produce antibody molecules byrecombinant means and thereby generate gene sequences that code forspecific amino acid sequences found in the polypeptide structure of theantibodies. Such antibodies can be produced by either cloning the genesequences encoding the polypeptide chains of said antibodies or bydirect synthesis of said polypeptide chains, with in vitro assembly ofthe synthesized chains to form active tetrameric (H₂L₂) structures withaffinity for specific epitopes and antigenic determinants. This haspermitted the ready production of antibodies having sequencescharacteristic of neutralizing antibodies from different species andsources.

Regardless of the source of the antibodies, or how they arerecombinantly constructed, or how they are synthesized, in vitro or invivo, using transgenic animals, such as cows, goats and sheep, usinglarge cell cultures of laboratory or commercial size, in bioreactors orby direct chemical synthesis employing no living organisms at any stageof the process, all antibodies have a similar overall 3 dimensionalstructure. This structure is often given as H₂L₂ and refers to the factthat antibodies commonly comprise 2 light (L) amino acid chains and 2heavy (H) amino acid chains. Both chains have regions capable ofinteracting with a structurally complementary antigenic target. Theregions interacting with the target are referred to as “variable” or “V”regions and are characterized by differences in amino acid sequence fromantibodies of different antigenic specificity.

The variable regions of either H or L chains contains the amino acidsequences capable of specifically binding to antigenic targets. Withinthese sequences are smaller sequences dubbed “hypervariable” because oftheir extreme variability between antibodies of differing specificity.Such hypervariable regions are also referred to as “complementaritydetermining regions” or “CDR” regions. These CDR regions account for thebasic specificity of the antibody for a particular antigenic determinantstructure.

The CDRs represent non-contiguous stretches of amino acids within thevariable regions but, regardless of species, the positional locations ofthese critical amino acid sequences within the variable heavy and lightchain regions have been found to have similar locations within the aminoacid sequences of the variable chains. The variable heavy and lightchains of all antibodies each have 3 CDR regions, each non-contiguouswith the others (termed L1, L2, L3, H1, H2, H3) for the respective light(L) and heavy (H) chains. The accepted CDR regions have been describedby Kabat et al., J. Biol. Chem. 252:6609-6616 (1977).

In all mammalian species, antibody polypeptides contain constant (i.e.,highly conserved) and variable regions, and, within the latter, thereare the CDRs and the so-called “framework regions” made up of amino acidsequences within the variable region of the heavy or light chain butoutside the CDRs.

The antibodies disclosed according to the invention may also be whollysynthetic, wherein the polypeptide chains of the antibodies aresynthesized and, possibly, optimized for binding to the polypeptidesdisclosed herein as being receptors. Such antibodies may be chimeric orhumanized antibodies and may be fully tetrameric in structure, or may bedimeric and comprise only a single heavy and a single light chain. Suchantibodies may also include fragments, such as Fab and F(ab₂)′fragments, capable of reacting with and binding to any of thepolypeptides disclosed herein as being receptors.

In one aspect, the present invention relates to immunoglobulins, orantibodies, as described herein, that react with, especially where theyare specific for, the polypeptides having amino acid sequences asdisclosed herein, preferably those having an amino acid sequence of oneof SEQ ID NO: 5 and 6. Such antibodies may commonly be in the form of acomposition, especially a pharmaceutical composition. Such antibodies,by themselves, may have therapeutic value in that they are able to bindto, and thereby tie up, surface sites on cancerous cells. Where suchsites have some type of function to perform (i.e., where they aresurface enzymes, or channel structures, or structures that otherwisefacilitate, actively or passively, the transport of nutrients and othervital materials to the cell. Such nutrients serve to facilitate thegrowth and replication of the cell and molecules that bind to such sitesand thereby interfere with such activities can prove to have atherapeutic effect in that the result of such binding is to removesources of nutrients from such cells, thereby interfering with growthand replication. In like manner, such binding may serve to remove vitalenzyme activities from the cell's functional repertoire, thereby alsointerfering with viability and/or the ability of the cell to multiply ormetastasize. In addition, by binding to such surface sites, theantibodies may serve to prevent the cells from reacting to environmentalagents, such as cytokines and the like, that may facilitate growth,replication and metastasis, thereby further reducing the cancerousstatus of such cell and ameliorating the cancerous condition in apatient, even without proving fatal to the cell or cells so affected.

The methods of the present invention also include processes wherein thecancer cell is contacted in vivo as well as ex vivo with an agent thatcomprises a portion, or is part of an overall molecular structure,having affinity for an expression product of a gene corresponding to apolynucleotide sequence as disclosed herein, preferably where theexpression product is a cell surface structure, most preferably apolypeptide as disclosed herein, such as one that comprises an aminoacid sequence of SEQ ID NO: 5 and 6. In one such embodiment, saidportion having affinity for said expression product is an antibody,especially where said expression product is a polypeptide oroligopeptide or comprises an oligopeptide portion, or comprises apolypeptide.

In another aspect, the present invention also relates to an antibodythat reacts with a polypeptide as disclosed herein, preferably apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 5 and 6. Such an antibody may be polyclonal,monoclonal, recombinant or synthetic in origin. In one such embodiment,said antibody is associated, either covalently or non-covalently, with acytotoxic agent, for example, an apoptotic agent. It is thuscontemplated that the antibody acts a targeted vector for guiding anassociated therapeutic agent to a cancerous cell, such as a cellexpressing a polypeptide homologous to, if not identical to, apolypeptide as disclosed herein.

Where the cytotoxic agent is itself a polypeptide, said may be linkeddirectly to an antibody specific for a surface target on a cancer cell,such as where the polypeptide represents an extension of the amino acidchain of the antibody. In alternative embodiments, such molecules may becovalently linked through a linker sequence of long or short duration,such as an amino acid sequence of 5 to 10 residues in length. Where thecytotoxic agents is some small organic molecule, such as a small organiccompound, or some type of apoptotic agent, this may be covalently bondedto the antibody molecule or may be attached by some other type ofnon-covalent linkage, including hydrophobic and electrostatic linkages.Methods for forming such linkages, especially covalent linkages, arewell known to those skilled in the art.

The antibodies disclosed herein may also serve as targeting vectors formuch larger structures, such as liposomes. In one such embodiment, anantibody is part of, or otherwise linked to, or associated with, amembranous structure, preferably a liposome or possibly some type ofcellular organelle, which acts as a reservoir for a cytotoxic agent,such as ricin. The antibody then acts to target said liposome to acancerous tissue in an animal, whereupon the liposome provides a sourceof cytotoxic agents for localized treatment of a solid tumor or othertype of neoplasm.

The present invention further encompasses an immunogenic compositioncomprising a polypeptide disclosed herein, as well as compositionsformed using antibodies specific for these polypeptides.

Methods well known in the art for making formulations are found in, forexample, Remington: The Science and Practice of Pharmacy, (19th ed.) Ed.A. R. Gennaro, 1995, Mack Publishing Company, Easton, Pa. Formulationsfor parenteral administration may, for example, contain excipients,sterile water, or saline, polyalkylene glycols such as polyethyleneglycol, oils of vegetable origin, or hydrogenated napthalenes.Biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be used tocontrol the release of the compounds. Other potentially usefulparenteral delivery systems for agonists of the invention includeethylenevinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, and liposomes. Formulations for inhalation may containexcipients, or example, lactose, or may be aqueous solutions containing,for example, polyoxyethylene-9-lauryl ether, glycocholate anddeoxycholate, or may be oily solutions for administration in the form ofnasal drops, or as a gel. It should be noted that, where the therapeuticagent to be administered is an immunoconjugate, these sometimes containchemical linkages that are somewhat labile in aqueous media and thereformust be stored prior to administration is a more stable environment,such as in the form of a lyophilized powder.

Such an agent can be a single molecular structure, comprising bothaffinity portion and anti-cancer activity portions, wherein saidportions are derived from separate molecules, or molecular structures,possessing such activity when separated and wherein such agent has beenformed by combining said portions into one larger molecular structure,such as where said portions are combined into the form of an adduct.Said anti-cancer and affinity portions may be joined covalently, such asin the form of a single polypeptide, or polypeptide-like, structure ormay be joined non-covalently, such as by hydrophobic or electrostaticinteractions, such structures having been formed by means well known inthe chemical arts. Alternatively, the anti-cancer and affinity portionsmay be formed from separate domains of a single molecule that exhibits,as part of the same chemical structure, more than one activity whereinone of the activities is against cancer cells, or tumor formation orgrowth, and the other activity is affinity for an expression productproduced by expression of genes related to the cancerous process orcondition.

In one embodiment of the present invention, a chemical agent, such as aprotein or other polypeptide, is joined to an agent, such as anantibody, having affinity for an expression product of a cancerous cell,such as a polypeptide or protein encoded by a gene related to thecancerous process, preferably a gene as disclosed herein according tothe present invention, most preferably a polypeptide sequence disclosedherein. Thus, where the presence of said expression product is essentialto tumor initiation and/or growth, binding of said agent to saidexpression product will have the effect of negating said tumor promotingactivity. In one such embodiment, said agent is an apoptosis-inducingagent that induces cell suicide, thereby killing the cancer cell andhalting tumor growth.

Other genes within the cancer cell that are regulated in a mannersimilar to that of the genes disclosed herein and thus change theirexpression in a coordinated way in response to chemical compoundsrepresent genes that are located within a common metabolic, signaling,physiological, or functional pathway so that by analyzing andidentifying such commonly regulated groups of genes (groups that includethe gene, or similar sequences, disclosed according to the invention,one can (a) assign known genes and novel genes to specific pathways and(b) identify specific functions and functional roles for novel genesthat are grouped into pathways with genes for which their functions arealready characterized or described. For example, one might identify agroup of 10 genes, at least one of which is the gene as disclosedherein, that change expression in a coordinated fashion and for whichthe function of one, such as the polypeptide encoded by the sequencedisclosed herein, is known then the other genes are thereby implicatedin a similar function or pathway and may thus play a role in thecancer-initiating or cancer-facilitating process. In the same way, if agene were found in normal cells but not in cancer cells, or happens tobe expressed at a higher level in normal as opposed to cancer cells,then a similar conclusion may be drawn as to its involvement in cancer,or other diseases. Therefore, the processes disclosed according to thepresent invention at once provide a novel means of assigning function togenes, i.e. a novel method of functional genomics, and a means foridentifying chemical compounds that have potential therapeutic effectson specific cellular pathways. Such chemical compounds may havetherapeutic relevance to a variety of diseases outside of cancer aswell, in cases where such diseases are known or are demonstrated toinvolve the specific cellular pathway that is affected.

The polypeptides disclosed herein, preferably those of SEQ ID NO: 5 and6, also find use as vaccines in that, where the polypeptide represents asurface protein present on a cancer cell, such polypeptide may beadministered to an animal, especially a human being, for purposes ofactivating cytotoxic T lymphocytes (CTLs) that will be specific for, andact to lyze, cancer cells in said animal. Where used as vaccines, suchpolypeptides are present in the form of a pharmaceutical composition.The present invention may also employ polypeptides that have the same,or similar, immunogenic character as the polypeptides of SEQ ID NO: 5and 6 and thereby elicit the same, or similar, immunogenic responseafter administration to an animal, such as an animal at risk ofdeveloping cancer, or afflicted therewith. Thus, the polypeptidesdisclosed according to the invention will commonly find use asimmunogenic compositions.

Expression of a gene corresponding to a polynucleotide disclosed herein,when in normal tissues, may indicate a predisposition towardsdevelopment of breast or endometrium cancer. The encoded polypeptidemight then present a potentially useful cell surface target fortherapeutic molecules such as cytolytic antibodies, or antibodiesattached to cytotoxic, or cytolytic, agents.

The present invention specifically contemplates use of antibodiesagainst the polypeptides encoded by the polynucleotides corresponding tothe genes disclosed herein, whereby said antibodies are conjugates toone or more cytotoxic agents so that the antibodies serve to target theconjugated immunotoxins to a region of cancerous activity, such as asolid tumor. For many known cytotoxic agents, lack of selectivity haspresented a drawback to their use as therapeutic agents in the treatmentof malignancies. For example, the class of two-chain toxins, consistingof a binding subunit (or B-chain) linked to a toxic subunit (A-chain)are extremely cytotoxic. Thus, such agents as ricin, a protein isolatedfrom castor beans, kills cells at very low concentrations (even lessthan 10⁻¹¹ M) by inactivating ribosomes in said cells (see, for example,Lord et al., Ricin: structure, mode of action, and some currentapplications. Faseb J, 8: 201-208 (1994), and Blättler et al., Realizingthe full potential of immunotoxins. Cancer Cells, 1: 50-55 (1989)).While isolated A-chains of protein toxins that functionally resemblericin A-chain are only weakly cytotoxic for intact cells (in theconcentration range of 10⁻⁷ to 10⁻⁶ M), they are very potent cytotoxicagents inside the cells. Thus, a single molecule of the A-subunit ofdiphtheria toxin can kill a cell once inside (see: Yamaizumi et al., Onemolecule of diphtheria toxin fragment A introduced into a cell can killthe cell. Cell, 15: 245-250, 1978).

The present invention solves this selectivity problem by usingantibodies specific for antigens present on cancer cells to target thecytotoxins to said cells. In addition, use of antibodies decreasestoxicity because the antibodies are non-toxic until they reach the tumorand, because the cytotoxin is bound to the antibody, it is presentedwith less opportunity to cause damage to non-targeted tissues.

In addition, use of such antibodies alone can provide therapeuticeffects on the tumor through the antibody-dependent cellular cytotoxicresponse (ADCC) and complement-mediated cell lysis mechanisms.

A number of recombinant immunotoxins (for example, consisting of Fvregions of cancer specific antibodies fused to truncated bacterialtoxins) are well known (see, for example, Smyth et al., Specifictargeting of chlorambucil to tumors with the use of monoclonalantibodies, J. Natl. Cancer Inst., 76(3):503-510 (1986); Cho et al.,Single-chain Fv/folate conjugates mediate efficient lysis offolate-receptor-positive tumor cells, Bioconjug. Chem., 8(3):338-346(1997)). As noted in the literature, these may contain, for example, atruncated version of Pseudomonas exotoxin as a toxic moiety but thetoxin is modified in such a manner that by itself it does not bind tonormal human cells, but it retains all other functions of cytotoxicity.Here, recombinant antibody fragments target the modified toxin to cancercells which are killed, such as by direct inhibition of proteinsynthesis, or by concomitant induction of apoptosis. Cells that are notrecognized by the antibody fragment, because they do not carry thecancer antigen, are not affected. Good activity and specificity has beenobserved for many recombinant immunotoxins in in vitro assays usingcultured cancer cells as well as in animal tumor models. Ongoingclinical trials provide examples where the promising pre-clinical datacorrelate with successful results in experimental cancer therapy. (see,for example, Brinkmann U., Recombinant antibody fragments andimmunotoxin fusions for cancer therapy, In Vivo (2000) 14:21-27).

While the safety of employing immunoconjugates in humans has beenestablished, in vivo therapeutic results have been less impressive.Because clinical use of mouse MAbs in humans is limited by thedevelopment of a foreign anti-globulin immune response by the humanhost, genetically engineered chimeric human-mouse MAbs have beendeveloped by replacing the mouse Fc region with the human constantregion. In other cases, the mouse antibodies have been “humanized” byreplacing the framework regions of variable domains of rodent antibodiesby their human equivalents. Such humanized and engineered antibodies caneven be structurally arranged to have specificities and effectorfunctions determined by design and which characteristics do not appearin nature. The development of bispecific antibodies, having differentbinding ends so that more than one antigenic site can be bound, haveproven useful in targeting cancer cells. Thus, such antibody specificityhas been improved by chemical coupling to various agents such asbacterial or plant toxins, radionuclides or cytotoxic drugs and otheragents. (see, for example, Bodey, B. et al). Genetically engineeredmonoclonal antibodies for direct anti-neoplastic treatment and cancercell specific delivery of chemotherapeutic agents. Curr Pharm Des (2000)February;6(3):261-76). See also, Garnett, M. C., Targeted drugconjugates: principles and progress. Adv. Drug Deliv. Rev. (2001 Dec.17) 53(2):171-216; Brinkmann et al., Recombinant immunotoxins for cancertherapy. Expert Opin Biol Ther. (2001) 1(4):693-702.

Among the cytotoxic agents specifically contemplated for use asimmunoconjugates according to the present invention are Calicheamicin, ahighly toxic enediyne antibiotic isolated from Micromonosporaechinospora ssp. Calichensis, and which binds to the minor groove of DNAto induce double strand breaks and cell death (see: Lee et al.,Calicheamicins, a novel family of antitumor antibiotics. 1. Chemistryand partial structure of calichemicin g₁ . J Am Chem Soc, 109: 3464-3466(1987); Zein et al., Calicheamicin gamma 1I: an antitumor antibioticthat cleaves double-stranded DNA site specifically, Science, 240:1198-1201 (1988)). Useful derivatives of the calicheamicins includemylotarg and 138H11-Camθ. Mylotarg is an immunoconjugate of a humanizedanti-CD33 antibody (CD33 being found in leukemic cells of most patientswith acute myeloid leukemia) and N-acetyl gamma colicheamicin dimethylhydrazide, the latter of which is readily coupled to an antibody of thepresent invention (in place of the anti-CD33 but which can also behumanized by substitution of human framework regions into the antibodyduring production as described elsewhere herein) to form animmunoconjugate of the invention. (see: Hamann et al. GemtuzumabOzogamicin, A Potent and Selective Anti-CD33 Antibody-CalicheamicinConjugate for Treatment of Acute Myeloid Leukemia, Bioconjug. Chem. 13,47-58 (2002)) For use with 138H11-Camθ, 138H11 is an anti-γ-glutamyltransferase antibody coupled to theta calicheamicin through a disulfidelinkage and found useful in vitro against cultured renal cell carcinomacells. (see: Knoll et al., Targeted therapy of experimental renal cellcarcinoma with a novel conjugate of monoclonal antibody 138H11 andcalicheamicin θ₁ ^(I) , Cancer Res, 60: 6089-6094 (2000) The samelinkage may be utilized to link this cytotoxic agent to an antibody ofthe present invention, thereby forming a targeting structure for breastor endometrium cancer cells.

Also useful in forming the immunoconjugates of the invention is DC1, adisulfide-containing analog of adozelesin, that kills cells by bindingto the minor groove of DNA, followed by alkylation of adenine bases.Adozelesin is a structural analog of CC-1065, an anti-tumor antibioticisolated from microbial fermentation of Streptomyces zelensis, and isabout 1,000 fold more toxic to cultured cell lines that other DNAinteracting agents, such as cis-platin and doxorubicin. This agent isreadily linked to antibodies through the disulfide bond of adozelesin.(see: Chari et al., Enhancement of the selectivity and antitumorefficacy of a CC-1065 analogue through immunoconjugate formation, CancerRes, 55: 4079-4084 (1995)).

Maytansine, a highly cytotoxic microtubular inhibitor isolated from theshrub Maytenus serrata found to have little value in human clinicaltrials, is much more effective in its derivatized form, denoted DM1,containing a disulfide bond to facilitate linkage to antibodies, is upto 10-fold more cytotoxic (see: Chari et al., Immunoconjugatescontaining novel maytansinoids: promising anticancer drugs, Cancer Res,52: 127-131 (1992)). These same in vitro studies showed that up to fourDM1 molecules could be linked to a single immunoglobulin withoutdestroying the binding affinity. Such conjugates have been used againstbreast cancer antigens, such as the neu/HER2/erbB-2 antigen. (see:Goldmacher et al., Immunogen, Inc., (2002) in press); also see Liu, C.et al., Eradication of large colon tumor xenografts by targeted deliveryof maytansinoids, Proc. Natl. Acad. Sci. USA, 93, 8618-8623 (1996)). Forexample, Liu et al. (1996) describes formation of an immunoconjugate ofthe maytansinoid cytotoxin DM1 and C242 antibody, a murine IgG1immunoglobulin, available from Pharmacia and which has affinity for amucin-like glycoprotein variably expressed by human colorectal cancers.The latter immunoconjugate was prepared according to Chari et al.,Cancer Res., 52:127-131 (1992) and was found to be highly cytotoxicagainst cultured colon cancer cells as well as showing anti-tumoreffects in vivo in mice bearing subcutaneous COLO 205 human colon tumorxenografts using doses well below the maximum tolerated dose.

In addition, there are a variety of protein toxins (cytotoxic proteins),which include a number of different classes, such as those that inhibitprotein synthesis: ribosome-inactivating proteins of plant origin, suchas ricin, abrin, gelonin, and a number of others, and bacterial toxinssuch as pseudomonas exotoxin and diphtheria toxin.

Another useful class is the one including taxol, taxotere, and taxoids.Specific examples include paclitaxel (taxol), its analog docetaxel(taxotere), and derivatives thereof. The first two are clinical drugsused in treating a number of tumors while the taxoids act to induce celldeath by inhibiting the de-polymerization of tubulin. Such agents arereadily linked to antibodies through disulfide bonds withoutdisadvantageous effects on binding specificity.

In one instance, a truncated Pseudomonas exotoxin was fused to ananti-CD22 variable fragment and used successfully to treat patients withchemotherapy-resistant hairy-cell leukemia. (see: Kreitman et al.,Efficacy of the anti-CD22 recombinant immunotoxin BL22 inchemotherapy-resistant hairy-cell leukemia, N Engl J Med, 345: 241-247(2001)) Conversely, the cancer-linked peptides of the present inventionoffer the opportunity to prepare antibodies, recombinant or otherwise,against the appropriate antigens to target solid tumors, preferablythose of malignancies of breast or endometrium tissue, using the same orsimilar cytotoxic conjugates. Thus, many of the previously usedimmunoconjugates have been formed using antibodies against generalantigenic sites linked to cancers whereas the antibodies formed usingthe peptides disclosed herein are more specific and target theantibody-cytotoxic agent to a particular tissue or organ, thus furtherreducing toxicity and other undesirable side effects.

In addition, the immunoconjugates formed using the antibodies preparedagainst the cancer-linked antigens disclosed herein can be formed by anytype of chemical coupling. Thus, the cytotoxic agent of choice, alongwith the immunoglobulin, can be coupled by any type of chemical linkage,covalent or non-covalent, including electrostatic linkage, to form theimmunoconjugates of the present invention.

When used as immunoconjugates, the antitumor agents of the presentinvention represent a class of pro-drugs that are relatively non-toxicwhen first administered to an animal (due mostly to the stability of theimmunoconjugate), such as a human patient, but which are targeted by theconjugated immunoglobulin to a cancer cell where they then exhibit goodtoxicity. The tumor-related, associated, or linked, antigens, preferablythose presented herein, serve as targets for the antibodies (monoclonal,recombinant, and the like) specific for said antigens. The end result isthe release of active cytotoxic agent inside the cell after binding ofthe immunoglobulin portion of the immunoconjugate.

The cited references describe a number of useful procedures for thechemical linkage of cytotoxic agents to immunoglobulins and thedisclosures of all such references cited herein are hereby incorporatedby reference in their entirety. For other reviews see Ghetie et al.,Immunotoxins in the therapy of cancer: from bench to clinic, PharmacolTher, 63: 209-234 (1994), Pietersz et al. The use of monoclonal antibodyimmunoconjugates in cancer therapy, Adv Exp Med Biol, 353:169-179(1994), and Pietersz, G. A. The linkage of cytotoxic drugs to monoclonalantibodies for the treatment of cancer, Bioconjug Chem, 1:89-95 (1990).

Thus, the present invention provides highly useful cancer-associatedantigens for generation of antibodies for linkage to a number ofdifferent cytotoxic agents which are already known to have some in vitrotoxicity and possess chemical groups available for linkage toantibodies.

The present invention also relates to a process that comprises a methodfor producing a product, including the generation of test data,comprising identifying an agent according to one of the disclosedprocesses for identifying such an agent (i.e., the therapeutic agentsidentified according to the assay procedures disclosed herein) whereinsaid product is the data collected with respect to said agent as aresult of said identification process, or assay, and wherein said datais sufficient to convey the chemical character and/or structure and/orproperties of said agent. For example, the present inventionspecifically contemplates a situation whereby a user of an assay of theinvention may use the assay to screen for compounds having the desiredenzyme modulating activity and, having identified the compound, thenconveys that information (i.e., information as to structure, dosage,etc) to another user who then utilizes the information to reproduce theagent and administer it for therapeutic or research purposes accordingto the invention. For example, the user of the assay (user 1) may screena number of test compounds without knowing the structure or identity ofthe compounds (such as where a number of code numbers are used the firstuser is simply given samples labeled with said code numbers) and, afterperforming the screening process, using one or more assay processes ofthe present invention, then imparts to a second user (user 2), verballyor in writing or some equivalent fashion, sufficient information toidentify the compounds having a particular modulating activity (forexample, the code number with the corresponding results). Thistransmission of information from user 1 to user 2 is specificallycontemplated by the present invention.

It should be cautioned that, in carrying out the procedures of thepresent invention as disclosed herein, whether to form immunoconjugatesor screen for other antitumor agents using the genes and polypeptidesdisclosed herein, any reference to particular buffers, media, reagents,cells, culture conditions and the like are not intended to be limiting,but are to be read so as to include all related materials that one ofordinary skill in the art would recognize as being of interest or valuein the particular context in which that discussion is presented. Forexample, it is often possible to substitute one buffer system or culturemedium for another and still achieve similar, if not identical, results.Those of skill in the art will have sufficient knowledge of such systemsand methodologies so as to be able, without undue experimentation, tomake such substitutions as will optimally serve their purposes in usingthe methods and procedures disclosed herein.

The present invention will now be further described by way of thefollowing non-limiting example. In applying the disclosure of theexample, it should be kept clearly in mind that other and differentembodiments of the methods disclosed according to the present inventionwill no doubt suggest themselves to those of skill in the relevant art.The following example shows how a potential anti-neoplastic agent may beidentified using one or more of the genes disclosed herein.

EXAMPLE Determination of Gene Inhibitory Activity of an Anti-NeoplasticAgent

SW480 cells are grown to a density of 10 cells/cm² in Leibovitz's L-15medium supplemented with 2 mM L-glutamine (90%) and 10% fetal bovineserum. The cells are collected after treatment with 0.25% trypsin, 0.02%EDTA at 37° C. for 2 to 5 minutes. The trypsinized cells are thendiluted with 30 ml growth medium and plated at a density of 50,000 cellsper well in a 96 well plate (100 μl/well). The following day, cells aretreated with either compound buffer alone, or compound buffer containinga chemical agent to be tested, for 24 hours. The media is then removed,the cells lysed and the RNA recovered using the RNAeasy reagents andprotocol obtained from Qiagen. RNA is quantitated and 10 ng of sample in1 μl are added to 24 μl of Taqman reaction mix containing 1×PCR buffer,RNAsin, reverse transcriptase, nucleoside triphosphates, amplitaq gold,tween 20, glycerol, bovine serum albumin (BSA) and specific PCR primersand probes for a reference gene (18S RNA) and a test gene (Gene X).Reverse transcription is then carried out at 48° C. for 30 minutes. Thesample is then applied to a Perlin Elmer 7700 sequence detector and heatdenatured for 10 minutes at 95° C. Amplification is performed through 40cycles using 15 seconds annealing at 60° C. followed by a 60 secondextension at 72° C. and 30 second denaturation at 95° C. Data files arethen captured and the data analyzed with the appropriate baselinewindows and thresholds.

The quantitative difference between the target and reference gene isthen calculated and a relative expression value determined for all ofthe samples used. In this way, the ability of a chemotherapeutic agentto effectively and selectively reduce the activity of a cancer-specificgene is readily ascertained. The overall expression of thecancer-specific gene, as modulated by one chemical agent relative toanother, is also determined. Chemical agents having the most effect inreducing gene activity are thereby identified as the mostanti-neoplastic.

REFERENCES

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1. A process for identifying an agent that modulates the activity of acancer-related gene comprising: (a) contacting a compound with a cellcontaining a gene that corresponds to a polynucleotide having a sequenceselected from the group consisting of SEQ ID NO: 1, 2, 3 and 4 and underconditions promoting the expression of said gene; and (b) detecting adifference in expression of said gene relative to when said compound isnot present thereby identifying an agent that modulates the activity ofa cancer-related gene.
 2. The process of claim 1 wherein said gene has asequence selected from the group consisting of SEQ ID NO: 1, 2, 3 and 4.3. The process of claim 1 wherein the cell is a cancer cell and thedifference in expression is a decrease in expression.
 4. The process ofclaim 3 wherein said cancer cell is a breast or endometrium cancer cell.5. A process for identifying an anti-neoplastic agent comprisingcontacting a cell exhibiting neoplastic activity with a compound firstidentified as a cancer related gene modulator using the process of claim1 and detecting a decrease in said neoplastic activity after saidcontacting compared to when said contacting does not occur.
 6. Theprocess of claim 5 wherein said neoplastic activity is acceleratedcellular replication.
 7. The process of claim 5 wherein said decrease inneoplastic activity results from the death of the cell.
 8. A process foridentifying an anti-neoplastic agent comprising administering to ananimal exhibiting a cancer condition an effective amount of an agentfirst identified according to the process of claim 1 and detecting adecrease in said cancerous condition.
 9. A process for determining thecancerous status of a cell, comprising determining an increase in thelevel of expression in said cell of a gene that corresponds to apolynucleotide having a sequence selected from the group consisting ofSEQ ID NO: 1, 2, 3 and 4 wherein an elevated expression relative to aknown non-cancerous cell indicates a cancerous state or potentiallycancerous state.
 10. The process of claim 9 wherein said elevatedexpression is due to an increased copy number.
 11. An isolatedpolypeptide comprising an amino acid sequence homologous to an aminoacid sequence selected from the group consisting of SEQ ID NO: 5 and 6wherein any difference between said amino acid sequence and the sequenceof SEQ ID NO: 5 and 6 is due solely to conservative amino acidsubstitutions and wherein said isolated polypeptide comprises at leastone immunogenic fragment.
 12. An isolated polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 5and
 6. 13. An antibody that reacts with a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 5and
 6. 14. The antibody of claim 13 wherein said antibody is arecombinant antibody.
 15. The antibody of claim 13 wherein said antibodyis a synthetic antibody.
 16. The antibody of claim 13 wherein saidantibody is a humanized antibody.
 17. An immunoconjugate comprising theantibody of claim 13 and a cytotoxic agent.
 18. The antibody of claim 17wherein said cytotoxic agent is a member selected from the groupconsisting of a calicheamicin, a maytansinoid, an adozelesin, acytotoxic protein, a taxol, a taxotere, a taxoid and DC1.
 19. Theimmunoconjugate of claim 18 wherein said calicheamicin is calicheamicinγ₁ ^(I), N-acetyl gamma calicheamicin dimethyl hydrazide orcalicheamicin θ₁ ^(I).
 20. The immunoconjugate of claim 18 wherein saidmaytansinoid is DM1.
 21. The immunoconjugate of claim 18 wherein saidcytotoxic protein is ricin, abrin, gelonin, pseudomonas exotoxin ordiphtheria toxin.
 22. The immunoconjugate of claim 18 wherein said taxolis paclitaxel.
 23. The immunoconjugate of claim 18 wherein said taxotereis docetaxel.
 24. A process for treating cancer comprising contacting acancerous cell in vivo with an agent having activity against anexpression product encoded by a gene sequence selected from the groupconsisting of SEQ ID NO: 1, 2, 3 and
 4. 25. The process of claim 24wherein said agent is an antibody of claim
 13. 26. The process of claim24 wherein said agent is an immunoconjugate of claim
 17. 27. Animmunogenic composition comprising a polypeptide of claim
 11. 28. Animmunogenic composition comprising a polypeptide of claim
 12. 29. Theprocess of claim 24 wherein said cancer is breast or endometrium cancer.30. A process for treating cancer in an animal afflicted therewithcomprising administering to said animal an amount of an immunogeniccomposition of claim 27 sufficient to elicit the production of cytotoxicT lymphocytes specific for the polypeptide of claim
 11. 31. A processfor treating cancer in an animal afflicted therewith comprisingadministering to said animal an amount of an immunogenic composition ofclaim 28 sufficient to elicit the production of cytotoxic T lymphocytesspecific for the polypeptide of claim
 12. 32. A process for treating acancerous condition in an animal afflicted therewith comprisingadministering to said animal a therapeutically effective amount of anagent first identified as having anti-neoplastic activity using theprocess of claim
 8. 33. A process for protecting an animal againstcancer comprising administering to an animal at risk of developingcancer a therapeutically effective amount of an agent first identifiedas having anti-neoplastic activity using the process of claim
 8. 34. Theprocess of claim 30 wherein said animal is a human being.
 35. Theprocess of claim 30 wherein said cancer is breast or endometrium cancer.36. A method for producing test data with respect to the gene modulatingactivity of a compound comprising: (a) contacting a compound with a cellcontaining a polynucleotide comprising a nucleotide sequencecorresponding to a gene whose expression is increased in a cancerouscell over that in a non-cancerous cell and under conditions wherein saidpolynucleotide is being expressed, (b) determining a change inexpression of polynucleotides as a result of said contacting, and (c)producing test data with respect to the gene modulating activity of saidcompound based on a decrease in the expression of the determined genewhose expression is otherwise increased in a cancerous cell over that ina non-cancerous cell indicating gene modulating activity.